Digital light processing printed hydrogel scaffolds with adjustable modulus

Hydrogels are extensively explored as biomaterials for tissue scaffolds, and their controlled fabrication has been the subject of wide investigation. However, the tedious mechanical property adjusting process through formula control hindered their application for diverse tissue scaffolds. To overcome this limitation, we proposed a two-step process to realize simple adjustment of mechanical modulus over a broad range, by combining digital light processing (DLP) and post-processing steps. UV-curable hydrogels (polyacrylamide-alginate) are 3D printed via DLP, with the ability to create complex 3D patterns. Subsequent post-processing with Fe3+ ions bath induces secondary crosslinking of hydrogel scaffolds, tuning the modulus as required through soaking in solutions with different Fe3+ concentrations. This innovative two-step process offers high-precision (10 μm) and broad modulus adjusting capability (15.8–345 kPa), covering a broad range of tissues in the human body. As a practical demonstration, hydrogel scaffolds with tissue-mimicking patterns were printed for cultivating cardiac tissue and vascular scaffolds, which can effectively support tissue growth and induce tissue morphologies.


S1. Printability and Swelling Behaviors of the Hydrogel Formula
The utilization of digital light processing (DLP) technology in this study allowed for the efficient filling of voids with a hydrogel solution containing 4 wt% alginate, due to its optimal viscosity.The increased fabrication speed of the hydrogel sample enabled by the low viscosity of the solution helped mitigate damage caused by swelling during the printing process.Moreover, the viscosity of the hydrogel solution decreased with increasing temperature, and the viscosity of the 6 wt% hydrogel solution at 60°C was comparable to that of the 4 wt% hydrogel solution at room temperature.Although the 4 wt% hydrogel solution was selected as the final printing ink in this study, it should be noted that higher percentages of alginate could be manufactured at temperatures above ambient.The swelling behavior of hydrogel samples with varying alginate concentrations (0-6 wt%) was investigated in response to different Fe 3+ ion concentrations.As shown in Fig. S2, the deformation of hydrogel samples decreased with increasing alginate concentration.However, the effect of ion concentration on the deformation of the hydrogel samples became less pronounced as the ion concentration increased beyond 0.005M.This suggests that the alginate content is the primary factor affecting the deformation of hydrogel samples in an ion bath with Fe 3+ concentrations above 0.005M.

S2. Optimization of DLP Parameters
To determine the relationship between the depth of cure and energy density, the energy density was adjusted by controlling the exposure time at a fixed power (P = 43.1 mW/cm²).The upper surface of the channel was measured as the depth of cure corresponding to its specific energy density.When the exposure time was less than 9 seconds, the depth of cure was too shallow, causing the elastic force of the film to be insufficient to overcome the surface tension of the solution, preventing it from escaping from the channel surface (Fig. S3a).As a result, the thickness of such films could not be measured, and the curing depths of exposure times between 10 seconds and 20 seconds were used for the fitting curve.
Hydrogel samples were washed in a 40% ethanol solution for 15 minutes, followed by crosslinking with 0.1 M Fe 3+ .The stress-strain curves of the hydrogel samples before and after treatment showed no significant changes (Fig. S5a), indicating that brief ethanol solution washing did not disrupt the hydrogel sample.However, gelatin coating forms a polymer layer on the surface of the hydrogel samples.The interaction between gelatin and the hydrogel results in the formation of a semi-interpenetrating polymer network (semi-IPN), which improves the modulus (increase from 13 kPa to 17 kPa) of the non-crosslinked hydrogel (Fig. S5b).

S4. Patterned Tissue Induced by Hydrogel Scaffolds
Biocompatibility experiments were conducted using samples with varying degrees of crosslinking.Testing revealed that hydrogels crosslinked with Fe 3+ at concentrations ranging from 0 to 1M exhibited no significant toxicity compared to the control group (Fig. S6).This indicates that the PAAm-Alg hydrogel samples themselves are non-cytotoxic and that the system, following Fe 3+ crosslinking, remains relatively stable without causing substantial harm to the cells.Upon staining and observation, a distinct and organized tissue formation was evident within the microgrooves.Cardiac tissue aligned along the H-shaped grooves and the interconnectedness between parallel grooves was densely populated with cells, thus integrating the tissue into a cohesive whole (Fig. S8a-h).In contrast, cardiac tissue exhibited a fragmented structure on flat substrates, with clear boundaries between the individual clusters (Fig. S8i-p).
The OrientationJ plugin in ImageJ was used to quantify the orientation of cardiac tissue cultured on hydrogel scaffolds 1 .To compare the statistical data from different images (Fig. S7a, b), the data were normalized using the following formula 2 : where Intensity(x) is the normalized intensity at angle x, Dx is the measured value at angle x, and Dmin is the minimum value among the measured data.An angle of 90° corresponds to the orientation of the Hshaped grooves.Cardiac tissue cultured on micro-grooves hydrogel scaffolds showed significantly more alignment compared to those cultured on flat substrates.

Figure
Figure S1.a) the viscosity of hydrogel solution containing 4 wt%, 6 wt% alginate over a temperature range of 25℃-60℃ at 10 s -1 .b) the rheological properties of hydrogel solution containing 4 wt% alginate at room temperature compared to hydrogel solution containing 6 wt% alginate at 60°C.

Figure S3 .
Figure S3.Depth of cure of UV curable hydrogel solution at different exposure times @P=43.1 mW/cm 2 , the exposure times in Figure a-l correspond to 9-20s.

Figure S5 .
Figure S5.Impact of sample immersion in a 40 wt% ethanol solution and porcine gelatin.a) The stress-strain behavior of hydrogel samples with or without washing in 40 wt% ethanol solution followed by crosslinking in 0.1 M Fe 3+ ion baths.b) The stress-strain behavior of hydrogel samples before and after gelatin coating.

Figure S6 .
Figure S6.Biocompatibility experiments, the cell viability is tested by a CCK-8 kit and normalized by the value of the

Figure S7 .
Figure S7.Cardiac tissue directional analysis.a) The orientation of cardiac tissue on the hydrogel scaffold with microgrooves in relation to the number of days in culture.b) The orientation of cardiac tissue on flat hydrogel substance in relation to the number of days in culture.

Figure S8 .
Figure S8.After 3 days of culture, cardiac tissue was immunofluorescently stained on flexible scaffolds with periodic microgrooves (a-h) and flat substrates (i-p).Fluorescence signals showed GFP (green) from fluorescent cardiomyocytes, protein α-actinin (red), and cell nuclei (blue).d) Longitudinal and transverse microgrooves induced H-shaped connections within the tissue, maintaining organization and enhancing information transfer.h) Longitudinal grooves guided orderly cardiac cell arrangement.l, p) Cardiomyocytes cultured on flat substrates exhibited noticeable aggregation and distinct boundaries.

Figure S9 .
Figure S9.The original photo taken by Huawei Mate 30 mobile phone a) 3D octopus sample; b-c) Printed sample with double-helical flow channels